Although it is clear that alterations in mesenchymal or neuronal components of the digestive system play important roles in the pathophysiology of a number of congenital motility disorders of humans, little is known about the genetic regulation of the developmental interplay between these components, or about the genetic programs required for intestinal mesenchymal differentiation. Hlx, a gene encoding a homeobox transcription factor, is required for normal development and growth of the digestive system. We have found in preliminary experiments that Hlx is required for normal development of the enteric nervous system (ENS) and intestinal mesenchyme. Our overarching hypothesis is that the Hlx transcription factor regulates intestinal mesenchymal differentiation and mesenchymal-neuronal interactions required for ENS development. In this application, we propose three specific aims to understand the role of Hlx in ENS development and intestinal mesenchymal differentiation. First, we will test the hypothesis that Hlx is required for normal migration and intestinal colonization of neural crest cells in mouse development. For these experiments we will use the dopamine beta-hydroxylase-LacZ transgene to compare the position of neural crest cells in Hlx+/+ and Hlx-/- embryos. Our second aim is to test the hypothesis that Hlx is required for normal proliferation and/or differentiation of neural crest cells in mouse development. To test this hypothesis, we will use immunohistochemistry for specific enteric neuronal markers to assess the lineages of enteric neurons present in Hlx-/- as compare to Hlx+/+ embryos. Finally, we will test the hypothesis that Hlx is required for development of primitive intestinal mesenchymal cells into smooth muscle cells, interstitial cells of Cajal, and subepithelial myofibroblasts. We will use two complementary approaches to test this hypothesis: immunohistochemistry for markers of intestinal mesenchymal cell lineages in Hlx+/+ and Hlx-/- embryos, and transgene markers of smooth muscle differentiation (reporter proteins driven by alpha- smooth muscle actin and gamma-smooth muscle actin promoters). The proposed experiments will provide novel insights into the differentiation program of intestinal mesenchyme and enteric neurons and their regulation by the Hlx transcription factor. This research is complementary to separately funded research focusing on regulation of Hlx gene expression and the identification of downstream targets of the Hlx transcription factor, particularly those important for mesenchymal-epithelial interactions in development.